Journal:

Article Title: RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis

doi: 10.1093/nar/gkg768

Figure Lengend Snippet: Rapid disappearance of vigilin in cells transfected with vigilin-specific siRNA. (A) HeLa cells were either not transfected (Untrans), mock transfected (Mock), transfected with either of two vigilin-specific siRNAs (Vig 1 and Vig 2), or with a non-specific pGL3 luciferase-specific siRNA (pGL3). Forty-eight hours after transfection, the cells were harvested, extracts were prepared, and 10 µg of each sample was fractionated on an 8% polyacrylamide gel and analyzed for vigilin content by western blotting with polyclonal rabbit anti-vigilin antibody. Purified recombinant FLAG epitope-tagged vigilin (fVig) was run as a control. (B) HeLa cells were left untransfected (Untrans), mock transfected (Mock), or were transfected with Vig 2 siRNA (0.84 µg) or with increasing concentrations of control pGL3 siRNA (0.14–0.84 µg). Forty-eight hours post-transfection, cell extracts were prepared, and 10 µg of each sample was fractionated by SDS–PAGE and analyzed for vigilin by western blotting. (C) HeLa cells were transfected with pGL3 or Vig 2 siRNAs, cells were harvested at 12, 24, 36 and 48 h post-transfection, extracts were prepared, and 30 µg of each sample was fractionated on an 8% polyacrylamide gel and analyzed for vigilin by western blotting. As a control for the specificity of the siRNAs, 20 µg of each extract was run on a 12% SDS–PAGE gel and analyzed for actin content by western blotting.

Article Snippet: The membranes were blocked overnight at 4°C in 1% non-fat dry milk (1 h at room temperature in 5% non-fat dry milk for actin and PARP western blots), then probed with either rabbit polyclonal anti-vigilin, or anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-PARP (Cell Signaling, Beverly, MA, USA) antibody for 1 h at room temperature (overnight at 4°C for actin and PARP), washed and probed with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.

Techniques: Transfection, Luciferase, Western Blot, Purification, Recombinant, FLAG-tag, Control, SDS Page

Journal:

Article Title: RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis

doi: 10.1093/nar/gkg768

Figure Lengend Snippet: Vigilin knockdown in HeLa cells results in programmed cell death. HeLa cells were treated with 6.25 nM etoposide (a standard inducer of programmed cell death), mock transfected, or transfected with pGL3 or Vig 2 siRNAs. A western blot was performed 48 h post-transfection using a polyclonal antibody to poly(ADP-ribose) polymerase (PARP). A western blot using a polyclonal antibody to actin was also performed as a loading standard. Because the etoposide-treated cells were in the late stages of apoptosis, they exhibited reduced levels of actin. Levels of actin in the pGL3 and Vig 2-treated cells were similar and the ratio of the cleaved PARP to uncleaved PARP is dramatically increased in the Vig 2-treated cells relative to the mock or pGL3-treated cells.

Article Snippet: The membranes were blocked overnight at 4°C in 1% non-fat dry milk (1 h at room temperature in 5% non-fat dry milk for actin and PARP western blots), then probed with either rabbit polyclonal anti-vigilin, or anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-PARP (Cell Signaling, Beverly, MA, USA) antibody for 1 h at room temperature (overnight at 4°C for actin and PARP), washed and probed with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.

Techniques: Knockdown, Transfection, Western Blot

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a , Genome-scale CRISPR knockout screens for all four DENV serotypes (DENV-1 276RKI , DENV-2 429557 , DENV-3 Philippines/H871856 , and DENV-4 BC287/97 ) in Huh7.5.1 cells. The genetic screens were independently performed for each serotype, analyzed with MAGeCK, and combined to obtain significance scores (y-axis). The 50 most enriched genes were colored and grouped by function. b , Scatter plot depicting enrichment scores of high-confidence ChIRP-MS DENV hits (x-axis) and the 200 top scoring hits from DENV CRISPR genetic screens (y-axis). Common hits shared by both DENV genetic screens and DENV ChIRP-MS were colored in red (vigilin), blue (RRBP1), and purple (others). c , Western blot (WB) analysis of wild-type (WT) or clonal RRBP1 knock-out (KO) (upper panel) and vigilin-KO (lower panel) cells in Huh7.5.1 cells. Representative WB of n = 2 biologically independent replicates showing similar results. d , qRT-PCR analysis of DENV infected WT and RRBP1-KO Huh7.5.1 cells (48 hours post-infection (hpi), MOI of 0.1) or ZIKV PRVABC59 , POWV LB (48 hpi, MOI 0.1) and CHIKV 18½5 (24 hpi, MOI of 0.01) infected WT and RRBP1-KO Huh7.5.1 cells. e , qRT-PCR analysis as in ( d ) here with vigilin-KO. Note: The WT datasets for POWV in panel d and e derived from the same experiments. f , WB analysis of DENV-2 429557 infected (MOI of 0.1, 72 hpi) WT, RRBP1-KO, and vigilin-KO Huh7.5.1 cell lysates, probed with DENV prM and NS3 antibodies. Representative WB of n = 4 biologically independent replicates showing similar results. g , Titers of infectious particles production from WT, RRBP1-KO, and vigilin-KO Huh7.5.1 cells infected with DENV-2 429557 at MOI of 0.1 for 72 h. For panel d , e , and g, the datasets represent the mean with standard error of the mean (SEM) of n = 3 independent biological replicates, except for POWV, n = 4 independent biological replicates. All P -values were determined by two-tailed, unpaired t -test using GraphPad Prism (GraphPad Software), where * = P <0.05 and n.s. = non-significant.

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: CRISPR, Knock-Out, Western Blot, Quantitative RT-PCR, Infection, Derivative Assay, Two Tailed Test, Software

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a, Single cell quantification and correlation between, RRBP1, vigilin, ER-GFP (an ER marker), and 4′,6-diamidino-2-phenylindole (DAPI) immunofluorescence signals. Total number of cells that were randomly chosen for each analysis, with mean and SEM are indicated. b , Western blot analysis of ER and cytosolic cell fractions probed with GAPDH or Tubulin (cytosolic markers), RPN1 (ER marker), RRBP1, and vigilin antibodies. Representative WB of n = 3 biologically independent replicates showing similar results. c , Western blot analysis of three independent co-IP experiments from non-infected Huh7.5.1 cells with RRBP1 as the bait showing similar results. Samples were treated with or without RNase A. d , and e , Representative IF of RRBP1 ( d ), vigilin ( e ) co-stained with RNA fluorescent in situ hybridization targeting (RNA-FISH) of DENV or ZIKV positive stranded RNA genomes. Representative images of n = 2 biologically independent replicates showing similar results. Scale bars, 10 μm.

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: Marker, Immunofluorescence, Western Blot, Co-Immunoprecipitation Assay, Infection, Staining, In Situ Hybridization

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a , RRBP1 (left) and vigilin (right) irCLIP reverse transcriptase (RT) stop mapping statistics annotated to the human, DENV, or ZIKV genomes and the ribosomal RNAs (rRNA) from Huh7.5.1 cells infected with MOI of 0.1 for 48 h. b , Histogram of RT stops mapping to the rRNAs from the RRBP1 (top) and vigilin (bottom) irCLIP in uninfected Huh7.5.1 cells. The three cytosolic rRNAs are highlighted. Red dashed line denotes vigilin’s strongest binding site, which is adjacent to RRBP1’s. c , Annotation of peaks called from RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapping to functional elements of human mRNAs including 5’UTR, exons, 3’UTR, and introns. Enrichment values are calculated based on the size of each function domain relative to the human genome. d , RRBP1 (top) and vigilin (bottom) irCLIP RT stops mapped at base resolution to the DENV genome. RT stop intensity was normalized to the total number of unique reads mapping to the viral genome. The 5’UTR and 3’UTR regions are highlighted in red and blue, respectively.

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: Reverse Transcription, Infection, Binding Assay, Functional Assay

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a , and b , Time-course DENV-Luc infection assays. WT, RRBP1-KO, and RRBP1-KO + RRBP1 cDNA rescue ( a ) or WT, vigilin-KO, and vigilin-KO + vigilin cDNA rescue ( b ) HEK293FT cells were infected with DENV-Luc (MOI of 0.01) and harvested at indicated time points. Virus infectivity was then determined by measuring Renilla luciferase expression from infected cells. c , and d , Time-course CVB3-Luc infection assays. WT, RRBP1-KO, and RRBP1-KO + RRBP1 cDNA rescue ( c ) or WT, vigilin-KO, and vigilin-KO + vigilin cDNA rescue ( d ) HEK293FT cells were infected with CVB3-Luc (MOI of 1) and harvested at indicated time points. e , and f , Luciferase expression of luciferase-encoding DENV replicon RNA in WT and RRBP1-KO ( e ) or WT and vigilin-KO ( f ) HEK293FT cells over indicated time points post-electroporation of replicon RNA. The data in each panel ( a - f ) represent the mean with SEM of of n = 3 independent biological replicates. g , Luciferase expression 8 hours (left) post-DENV-Luc (MOI of 0.025) infection in the presence of the replication inhibitor MK0608 (50 μM final concentration) or 36 hours (right) post DENV-Luc infection in the presence of DMSO of WT, RRBP1-KO, and vigilin-KO HEK293FT cells. The data in each panel represents the mean with SEM of 10 biologically independent infections. Fold change between datasets is indicated. All P -values stated in this figure were determined by two-tailed, unpaired t -test using GraphPad Prism, where * = P < 0.05 and n.s. = non-significant.

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: Infection, Virus, Luciferase, Expressing, Electroporation, Concentration Assay, Two Tailed Test

Journal: Nature microbiology

Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection

doi: 10.1038/s41564-019-0518-2

Figure Lengend Snippet: a , Western blot analysis of WT Huh7.5.1, vigilin-KO, RRBP1-KO, and RRBP1-vigilin double-KO cells. Representative WB of n = 2 biologically independent replicates showing similar results. b , Luciferase expression at 8 hpi and 24 hpi upon DENV-Luc infection (MOI 0.01) of WT Huh7.5.1, vigilin-KO, RRBP1-KO, and RRBP1-vigilin double-KO cells. The data in each panel represent the mean and SEM of 9 biologically independent infections. The P -values were determined by two-tailed, unpaired t -test using GraphPad Prism, where * = P < 0.05 and n.s. = non-significant. c , Northern blot analysis of dengue genomic RNA extracted from WT Huh7.5.1 and RRBP1-vigilin double-KO cells that were first infected with DENV-2 16681 (MOI of 0.1) for 48 hours, followed by MK0608 replication inhibitor treatment for indicated time frames (top panel). Quantification of DENV genomic RNA (i.e. northern blot signal) from 3 independent experiments (error bars are SEM) as a percentage relative to time point 0 hour after MK0608 treatment (bottom).

Article Snippet: Western blot analysis from the two different fractions was then performed with antibodies against RRBP1, vigilin, the cytoplasmic markers GAPDH (Genetex GTX 627408) and tubulin (Abcam ab97872) and the ER marker RPN1 (Bethyl Laboratories A305–026A).

Techniques: Western Blot, Luciferase, Expressing, Infection, Two Tailed Test, Northern Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Cold-Induced Lipoprotein Clearance in Cyp7b1 -Deficient Mice

doi: 10.3389/fcell.2022.836741

Figure Lengend Snippet: Cyp7b1 deletion impairs BAT lipoprotein processing responses during cold exposure. WT (n = 9) and Cyp7b1 −/− (n = 8) littermates were fed a WTD and housed for 1 week at a cold (6°C) environment. BAT relative gene expression of lipoprotein processing/uptake, glucose uptake, and de novo lipogenesis markers (A) , protein levels of LPL, GPIHBP1, FASN, p-AKT and AKT (B) with relative quantification of LPL, GPIHBP1, and FASN (C) . Plasma insulin levels (D) , relative quantification of p-AKT and AKT blots shown in B (E) , and HOMA-IR (F) . Data are shown as mean values ±SEM. Different letters indicate significant differences between groups ( p < 0.05) determined by two-way ANOVA.

Article Snippet: Quantitative real-time PCR was performed either on an ABI 7900HT or a QuantStudioTM 5 Real-Time PCR System (Applied Biosystems) using TaqMan on-demand primer sets (Applied Biosystems, Waltham, MA, United States) ( Acaca : Mm01304285_m1, Angptl4 : Mm00480431_m1, CD36 : Mm00432403_m1, Chrebp-beta : AIVI4CH, Cyp27a1 : Mm00470430_m1, Cyp7a1 : Mm00484150_m1, Cyp7b1 : Mm00484157_m1, Cyp8b1 : Mm00501637_s1, Fasn : Mm00662319_m1, Gpihbp1 : Mm01205849_g1, Lipe : Mm00495359_m1, Lipg : Mm00495368_m1, Lpl : Mm00434764_m1, Pnpla2 : Mm00503040_m1, Scd1: Mm00772290_m1, Slc27a1 : Mm00449511_m1, Slc27a4 : Mm01327405_m1, Slc27a5 : Mm00447768_m1, Slc2a1 : Mm00441480_m1, Slc2a4 : Mm01245502_m1). mRNA levels were normalized to the level of the housekeeping gene TATA-box binding protein (Tbp) mRNA, and the results were displayed as relative gene expression normalized to the experimental control group, following calculations using the 2 -ΔΔCt method.

Techniques: Gene Expression, Quantitative Proteomics, Clinical Proteomics

Journal: Journal of Neuroscience Research

Article Title: Neuronal extracellular microRNAs miR‐124 and miR‐9 mediate cell–cell communication between neurons and microglia

doi: 10.1002/jnr.24344

Figure Lengend Snippet: Antibody characterization

Article Snippet: Rabbit polyclonal antibodies for human/mouse/rat HDLs (immunogen: KLH conjugated synthetic peptide from a 41–125 amino acid sequence of human HDL) were purchased from Biorbyt (cat#orb1003; 0.5 mg/ml, dilution 1:1,000; RRID: AB_2737031).

Techniques: Concentration Assay, Marker, Binding Assay, Immunofluorescence, Blocking Assay, Sequencing, Western Blot